Methods Of Producing And Sequencing Modified Polynucleotides

ABSTRACT

The present invention encompasses methods for producing a modified polynucleotide sequence that comprises a (e.g., one or more) phosphorothiolate linkage, methods for determining a polynucleotide sequence comprising a (e.g., one or more) phosphorothiolate linkage, and methods for separating forward and reverse extension products that comprise a (e.g., one or more) phosphorothiolate linkage. The invention also encompasses kits for producing and/or determining the sequence of a modified polynucleotide that comprises a (e.g., one or more) phosphorothiolate linkage.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.11/476,423, filed Jun. 28, 2006, which claims the benefit of U.S.Provisional Application No. 60/694,783, filed on Jun. 28, 2005. Theentire teachings of the above applications are incorporated herein byreference.

GOVERNMENT SUPPORT

The invention was supported, in whole or in part, by a grant HG00357from the National Institutes of Health. The Government has certainrights in the invention.

BACKGROUND OF THE INVENTION

Widespread efforts have been made in recent years to determine thesequence of the human genome, as well as the genomes of various otherorganisms. The advent of genomics has relied upon accurate and efficientDNA sequencing techniques, and the ability to determine the nucleotidesequence of a gene remains an essential component of molecular geneticresearch. The widespread use of DNA sequencing in biological researchnecessitates the development of new DNA sequencing techniques that aresimpler and more efficient than traditional, commonly-used techniques.

Classical DNA sequencing techniques, such as the Sanger chaintermination method (Sanger, F., et al. Proc. Natl. Acad. Sci. USA 74:5463-5467 (1977); incorporated herein by reference) and the Maxam andGilbert chemical cleavage method (Maxam, A. M. and Gilbert, W. Proc.Natl. Acad. Sci. USA 74: 560-564 (1977); incorporated herein byreference), are somewhat cumbersome and inefficient, as both of theseapproaches require researchers to perform multiple reactions in order toderive a nucleotide sequence. Attempts to simplify DNA sequencing bycoupling a single DNA amplification/synthesis reaction with sequenceanalysis (i.e., direct sequencing) have shown limited success, becausethese techniques often result in DNA damage and/or degradation (Das etal., Physiol Genomics 6: 57-80 (2001)), as well as low fidelity DNAsynthesis (Lin et al., Biochemistry 40: 8749-8755 (2001); Xia et al.,Proc Natl Acad Sci USA 99: 6597-6602 (2002); U.S. Pat. No. 5,939,292;U.S. Pat. No. 6,329,178; and U.S. Pat. No. 6,887,690).

Presently, there is a clear need to develop improved methods ofsequencing DNA. More specifically, there is a need to develop reliableand efficient direct sequencing techniques that yield accurate DNAsequence information.

SUMMARY OF THE INVENTION

The invention of the instant application provides new methods ofsequencing nucleic acids (e.g., DNA), as well as improved methods forperforming direct nucleic acid (e.g., DNA) sequencing. The presentinvention is based, in part, on the discovery that nucleic acidpolymerases (e.g., DNA polymerases) are capable of incorporatingthiol-nucleoside triphosphates (e.g., 5′ thiol-nucleoside triphosphates,3′ thiol-nucleoside triphosphates) into a growing polynucleotide strandto generate a polynucleotide sequence that comprises at least onephosphorothiolate linkage. Accordingly, the invention encompasses amethod for producing a modified polynucleotide sequence that comprises a(one or more) phosphorothiolate linkage. The method comprises annealingat least one primer to a template polynucleotide sequence and extendingthe primer in the presence of one or more nucleoside triphosphates,wherein at least one of the nucleoside triphosphates is modified, suchthat a polynucleotide sequence that comprises a phosphorothiolatelinkage is produced.

In a particular embodiment, the invention provides a method forproducing a modified polynucleotide sequence that comprises a 3′phosphorothiolate linkage. The method comprises annealing at least oneprimer to a template polynucleotide sequence and extending said primerin the presence of one or more nucleoside triphosphates, wherein atleast one of the nucleoside triphosphates in the mixture is a modifiednucleoside triphosphate comprising the general structure [I]:

such that a polynucleotide sequence comprising at least one modifiednucleotide is produced. The modified polynucleotide sequence comprises a3′ phosphorothiolate linkage, illustrated by the general structure [II]:

R₁ in general structure [I] can be, for example, hydrogen (—H), asubstituted or non-substituted: alkyl, akenyl, alkynyl, or aryl group,or R₂, wherein R₂ can be, for example, —SH, or —SR₃, and wherein R₃ canbe, for example, a substituted or non-substituted: alkyl, akenyl,alkynyl, or aryl group. In a particular embodiment, R₁ is —SCH₃.

In another embodiment, the invention provides a method for producing amodified polynucleotide sequence that comprises a 5′ phosphorothiolatelinkage. The method comprises annealing at least one primer to atemplate polynucleotide sequence and extending said primer in thepresence of one or more nucleoside triphosphates, wherein at least oneof the nucleoside triphosphates is a modified nucleoside triphosphatecomprising the general structure [III]:

such that a modified polynucleotide sequence is produced. The modifiedpolynucleotide sequence comprises a 5′ phosphorothiolate linkage,illustrated by the general structure [IV]:

The present invention also encompasses a method for determining (e.g.,sequencing) a polynucleotide sequence comprising annealing a pluralityof primers to a plurality of template polynucleotide sequences andextending the plurality primers in the presence of nucleosidetriphosphates, wherein at least one of the nucleoside triphosphates ismodified, thereby producing a plurality of extension products thatcomprise a modified nucleotide sequence having one or morephosphorothiolate linkages. The phosphorothiolate linkages in theextension products are cleaved under conditions in which a plurality offragments are produced. The fragments that comprise the primer areidentified, and the nucleotide at the 3′ end of each fragment thatcomprises the primer is identified, such that the polynucleotidesequence can be determined.

For example, the fragments that comprise the primer can be identified(e.g., using a tag on the primer) and resolved (e.g., on a solidsupport, such as a gel), and the sequence of the polynucleotide can bedetermined (e.g., by detecting the length of the fragment for which thenucleotide at its 3′ end is known; by detecting a tag present on thenucleotide at the 3′ end, thereby identifying the nucleotide at the 3′end), as will be understood by one of skill in the art. The fragmentsattached to a primer can be identified either directly or indirectly,using one or more of a variety of previously-described techniques in theart, such as, for example, by using an isolating means that binds toand/or recognizes a tag on each primer. In the methods for determining apolynucleotide sequence, the phosphorothiolate linkage can be cleavedusing, for example, Ag⁺, Hg⁺⁺ and/or Cu⁺⁺. In particular embodiments,the methods comprise cleaving the phosphorothiolate linkage using Ag⁺ions at a pH of about 7.0 and at a temperature of about 22° C. to about37° C.

In a particular embodiment, the one or more modified nucleosidetriphosphates comprises a general structure [I]:

such that the modified nucleotide sequences that are produced comprise ageneral structure [II]:

wherein the general structure [II] comprises at least one 3′phosphorothiolate linkage.

R₁ in general structure [I] can be, for example, hydrogen (—H), asubstituted or non-substituted: alkyl, akenyl, alkynyl or aryl group, orR₂, wherein R₂ can be, for example, —SH, or —SR₃, and wherein R₃ can be,for example, a substituted or non-substituted: alkyl, akenyl, alkynyl,or aryl group. In a particular embodiment, R₁ is —SCH₃.

In another embodiment, the one or more nucleoside triphosphates comprisethe general structure [III]:

such that the modified nucleotide sequences that are produced comprise ageneral structure [IV]:

wherein the general structure [IV] comprises at least one 5′phosphorothiolate linkage.

In a further embodiment, the methods for determining a polynucleotidesequence also comprise isolating the cleaved fragments that comprise aprimer, prior to identifying the length of the polynucleotide, therebyidentifying the nucleotide at the 3′ end of the fragments by virtue ofthe modified nucleotide used in the extension reaction, or identifyingthe nucleotide at the 3′ end of the fragments by detecting a tag on thenucleotide. In a particular embodiment, the fragments that comprise aprimer are isolated using an isolating means that specificallyrecognizes a tag on the fragments (e.g., by binding of the isolatingmeans to a tag on each primer). As used herein, the term “isolatedfragment” refers to a preparation of fragments that is purified from, orotherwise substantially free of, other components from the extensionand/or cleavage reactions, including, but not limited to, cleavagefragments that are not attached to a primer, buffers, salts, metal ions,unincorporated nucleotides, nucleic acid templates and enzymes. The term“isolating means”, as used herein, refers to a means, such as a solidsupport, which comprises a moiety that specifically recognizes, andbinds to, a partner moiety on a substance to be isolated.

The invention is also directed to methods for separating one or moreforward extension products from one or more reverse extension products,comprising annealing a plurality of first primers and a plurality ofsecond primers to a plurality of template polynucleotide sequences thatcomprise a sense nucleotide strand and an antisense nucleotide strand,wherein the first primer anneals to the sense strand and the secondprimer anneals to the antisense strand and wherein at least one primer(e.g., the first primer, the second primer) comprises a tag. The firstand second primers are extended in the presence of one or morenucleoside triphosphates wherein at least one of the nucleosidetriphosphates is modified, thereby producing a plurality of extensionproducts that comprise a modified nucleotide sequence having one or morephosphorothiolate linkages. In particular, as will be understood by aperson of skill in the art, extension of a first primer annealed to asense nucleotide strand produces a reverse extension product andextension of a second primer annealed to an antisense nucleotide strandproduces a forward extension product. The phosphorothiolate linkages inthe modified nucleotide sequences are cleaved under conditions in whicha plurality of fragments of the reverse extension product and aplurality of fragments of the forward extension product are produced.The fragments of the reverse extension product that comprise the firstprimer are then separated from the fragments of the forward extensionproduct that comprise the second primer (e.g., using a tag on the firstand/or second primer), thereby separating forward and reverse extensionproducts. For example, one or more reverse extension products thatcomprise a first primer, which comprises a biotin tag, are separatedfrom fragments of the forward extension product that comprise the secondprimer, which do not comprise a biotin tag by binding to, for example,streptavidin.

In a particular embodiment, the first primer and the second primer eachcomprise a tag, wherein the tag on the first primer is distinct from thetag on the second primer. Accordingly, the fragments of the reverseextension product that comprise the first primer can be separated fromthe fragments of the forward extension product that comprise the secondprimer using the distinct tags on the first and second primers.

The instant invention also encompasses kits that comprise one or morenucleoside triphosphates, wherein at least one of the nucleosidetriphosphates is a modified thiol-nucleoside triphosphate, and a nucleicacid polymerase.

In a particular embodiment, the at least one modified thiol-nucleosidetriphosphate comprises the general structure [I]:

R₁ in general structure [I] can be, for example, hydrogen (—H), asubstituted or non-substituted: alkyl, akenyl, alkynyl or aryl group, orR₂, wherein R₂ can be, for example, —SH, or —SR₃, and wherein R₃ can be,for example, a substituted or non-substituted: alkyl, akenyl, alkynyl oraryl group. In a particular embodiment, R₁ is —SCH₃.

In another embodiment, the at least one modified thiol-nucleosidetriphosphate comprises the general structure [III]:

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A-FIG. 1D are schematics illustrating the steps involved inproducing, cleaving and isolating primer extension products, each ofwhich contains a labeled 3′ thiol nucleotide at its 3′ end.

FIG. 1A is a schematic depicting primer extension of a DNA templateusing a mixture of unmodified nucleoside triphosphates (pppN, alsoreferred to herein as dNTP) and modified 3′-thiol-nucleosidetriphosphates (pppAs, pppTs, pppGs, and pppCs, also referred to hereinas pppA-s, pppT-s, pppG-s, pppC-s, sdATP, sdTTP, sdGTP, sdCTP, dAsTP,dTsTP, dGsTP, dCsTP, As, Ts, Gs and Cs). Each of the four3′-thiol-nucleoside triphosphates is labeled with a distinctfluorophore, indicated by a colored star. The DNA primer (red) isattached to an affinity probe or tag, shown as a gray pentagonalstructure at the 5′ end of the primer.

FIG. 1B is a schematic depicting a DNA strand (top) which hasincorporated multiple labeled 3′-thiol nucleotides (As, Ts, Gs, Cs)following primer extension by DNA polymerase on a DNA template (bottomstrand).

FIG. 1C is a schematic depicting the selective cleavage of DNA strandscontaining 3′-thiol nucleotides in the presence of AgNO₃. Cleavageoccurs at the 3′ end of each modified 3′-thiol nucleotide and results inthe generation of several labeled cleavage products.

FIG. 1D is a schematic depicting the purification of several different5′ extension products following AgNO₃-induced cleavage of DNA strandscontaining 3′-thiol nucleotides. DNA cleavage products are isolatedusing a solid support (red circle), which contains a molecule (bluecross) that binds to the affinity tag on the primer (gray pentagon).Therefore, only the 5′-most DNA fragments, which comprise the primersequence, are recovered. All other labeled fragments are washed away.

FIG. 2A-FIG. 2C are schematics illustrating the steps involved inproducing, cleaving and separating forward and reverse primer extensionproducts, each of which contains a labeled 3′ thiol nucleotide at its 3′end.

FIG. 2A is a schematic depicting bidirectional PCR amplification of DNAusing a mixture of unmodified nucleoside triphosphates (pppN) andmodified 3′-thiol-nucleoside triphosphates (pppA-s, pppT-s, pppG-s, andpppC-s). The four 3′-thiol-nucleoside triphosphates are differentiallylabeled with distinct fluorophores, indicated by stars of differentcolors. The forward DNA primer (red) and reverse DNA primer (green) areattached to different affinity probes, shown as a gray pentagon (forwardprimer) or purple hexagon (reverse primer). The DNA duplex at the bottomof the Figure has incorporated multiple labeled 3′-thiol nucleotides(As, Ts, Gs, Cs) following primer extension by DNA polymerase.

FIG. 2B is a schematic depicting the selective cleavage of DNA strandscontaining 3′-thiol nucleotides in the presence of AgNO₃. Cleavageoccurs at the 3′ end of each modified 3′-thiol nucleotide and results inthe generation of several labeled cleavage products.

FIG. 2C is a schematic depicting the purification of 5′ extensionproducts following AgNO₃-induced cleavage of forward- and reverse-primedDNA strands containing 3′-thiol nucleotides. DNA cleavage productsgenerated from the forward-primed strand are isolated using a solidsupport (red circle), which contains a molecule (blue cross) that bindsto the affinity tag on the primer (gray pentagon). DNA cleavage productsgenerated from the reverse-primed strand are isolated using a solidsupport (red circle), which contains a molecule (blue sun) that binds tothe affinity tag on the reverse primer (purple hexagon). Therefore, the5′-most DNA fragments from both the forward and reverse strands can berecovered separately and analyzed to determine sequence.

FIG. 3A-FIG. 3E demonstrate the incorporation of 3′-deoxy-dithiomethylthymidine (dTsTP) into a growing DNA strand by a DNA polymerase.

FIG. 3A depicts the chemical structure of 3′-deoxy-dithiomethylthymidine (dTsTP).

FIG. 3B is a representation showing a dual biotin-labeled DNA templateattached to a magnetic bead. The template is hybridized to a primer,which is labeled with Cy5. Five adenine (A) nucleotides are locateddownstream of the portion of the template sequence that is complementaryto the primer sequence.

FIG. 3C depicts the fluorescence profile generated by the Cy5-labeledprimer, shown in FIG. 3B. Fluorescence intensity is indicated along theY-axis, while the size of the labeled product is indicated on theX-axis, where units 0, 1, 2, 3, 4 and 5, indicate that the primer hasbeen extended by 0, 1, 2, 3, 4, or 5 additional nucleotides,respectively. Fluorescence resulting from the label on the primer isindicated by a blue peak. Orange peaks represent the fluorescent profileof size standards.

FIG. 3D depicts the fluorescence profile of the DNA extension productsgenerated when DNA synthesis is conducted in the presence of3′-deoxy-dithiomethyl thymidine (dTTP) at a 5 μM concentration.Fluorescence intensity is indicated along the Y-axis, while the size ofthe labeled product is indicated on the X-axis, where units 0, 1, 2, 3,4 and 5, indicate that the primer has incorporated 0, 1, 2, 3, 4, or 5additional nucleotides, respectively. Fluorescence resulting from thelabel on the primer is indicated by a blue peak. The products from thisreaction contain anywhere from 0 to 5 modified thymidine residues, withthe majority of products containing either 0 or 1 modified nucleotides.Orange peaks represent the fluorescent profile of size standards.

FIG. 3E depicts the fluorescence profile of the DNA extension productsgenerated when DNA synthesis is conducted in the presence of 1.0 mM3′-deoxy-dithiomethyl thymidine (dTsTP). Fluorescence intensity isindicated along the Y-axis, while the size of the labeled product isindicated on the X-axis, where units 0, 1, 2, 3, 4 and 5 indicate thatthe primer has incorporated 0, 1, 2, 3, 4, or 5 additional nucleotides,respectively. Fluorescence resulting from the label on the primer isindicated by a blue peak. Nearly all of the products from this reactionhave incorporated 5 modified thymidine nucleotides. Orange peaksrepresent the fluorescent profile of size standards.

FIG. 4A-FIG. 4E depict incorporation of 3′-deoxy-dithiomethyl thymidine

(dTsTP) nucleotides into primer extension products, followed bysubsequent cleavage of the nucleotides in the presence of silver.

FIG. 4A depicts the fluorescence profile (blue) of uncleaved primerextension products generated in the presence of 50 μM3′-deoxy-dithiomethyl thymidine (dTsTP) nucleoside triphosphates.

FIG. 4B depicts the fluorescence profile of cleaved primer extensionproducts (blue peaks), which were generated in the presence of 50 μM3′-deoxy-dithiomethyl thymidine (dTsTP) nucleoside triphosphates,following treatment with AgNO₃. Orange peaks represent the fluorescentprofile of size standards.

FIG. 4C depicts the fluorescence profile (blue) of uncleaved primerextension products generated in the presence of 500 μM3′-deoxy-dithiomethyl thymidine (dTTP) nucleoside triphosphates.

FIG. 4D depicts the fluorescence profile of cleaved primer extensionproducts (blue peaks), which were generated in the presence of 500 μM3′-deoxy-dithiomethyl thymidine (dTsTP) nucleoside triphosphates,following treatment with AgNO₃. Orange peaks represent the fluorescentprofile of size standards.

FIG. 4E depicts the fluorescence profile generated by the labeled-primeralone. Fluorescence resulting from the label on the primer is indicatedby a blue peak. Orange peaks represent the fluorescent profile of sizestandards.

FIG. 5 depicts the chemical structure of a polynucleotide that comprisesa 3′ phosphorothiolate linkage.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on Applicants' discovery thatnucleic acid polymerases can incorporate thiol-nucleoside triphosphatesinto a growing polynucleotide strand to generate a polynucleotide thatcomprises one or more phosphorothiolate linkages. Thus, a polynucleotidecomprising a phosphorothiolate linkage can be enzymatically synthesizedusing a polymerase, rather than chemically synthesized. Therefore, thepresent invention provides a method for producing a modifiedpolynucleotide sequence, wherein the modified polynucleotide sequencecomprises a (one or more) scissile internucleoside linkage. As usedherein, a “scissile internucleoside linkage” or “scissile linkage”refers to an internucleoside linkage that can be cleaved underconditions that will not substantially cleave phosphodiester bonds. Asdescribed herein, nucleic acid polymerases can be used to generate apolynucleotide sequence that comprises one or more scissileinternucleoside linkage(s). In a particular embodiment, the scissileinternucleoside linkage is a (one or more) phosphorothiolate linkage.

Accordingly, the present invention provides methods for producing amodified polynucleotide sequence. As used herein, the term “modifiedpolynucleotide sequence” refers to a polynucleotide sequence thatcomprises one or more phosphorothiolate linkages. The method comprisesannealing at least one primer to a template polynucleotide sequence andextending the primer in the presence of one or more nucleosidetriphosphates, wherein at least one nucleoside triphosphate is modified.As used herein, the phrase “modified nucleoside triphosphate” or“nucleoside triphosphate that is modified” refers to a thiol-nucleosidetriphosphate that can be incorporated into a nascent polynucleotide by anucleic acid polymerase, thereby producing a modified polynucleotidesequence that comprises one or more phosphorothiolate linkages. The term“phosphorothiolate linkage”, as used herein, refers to a covalent bondbetween a sulfur and a phosphorus atom. As used herein, the phrase“polynucleotide sequence comprising a phosphorothiolate linkage” refersto a polynucleotide sequence that comprises a sulfur-phosphorus covalentbond. In particular, a phosphorothiolate linkage results when a sulfuratom replaces one of the bridging oxygen atoms in a phosphodiester bond.

In a particular embodiment, the polynucleotide sequence comprises a 3′phosphorothiolate linkage and is represented by the general structure[II]:

A modified polynucleotide sequence comprising a 3′ phosphorothiolatelinkage can be generated by incorporating one or more 3′thiol-nucleoside triphosphates into a growing polynucleotide strand. Asused herein, a “3′ thiol-nucleoside triphosphate” refers to a moleculehaving the general structure [I]:

-   -   wherein R₁ is hydrogen, a substituted or non-substituted: alkyl,        akenyl, alkynyl or aryl group, or R₂;    -   wherein R₂ is —SH, or —SR₃; and    -   wherein R₃ is a substituted or non-substituted: alkyl, akenyl,        alkynyl or aryl group.

As used herein, “alkyl”, “alkenyl” and “alkynyl” means a group that issaturated or unsaturated, straight-chain, branched, or cyclic, and isderived from a hydrocarbon radical derived by the removal of onehydrogen atom from a single carbon atom of a parent alkane, alkene, oralkyne, respectively. Typical alkyl (e.g., alkyl, cycloalkyl,heteroalkyl), alkenyl (e.g., alkenyl, cycloalkenyl, heteroalkenyl) andalkynyl (e.g., alkynyl, cycloalkynyl, heteroalkynyl) groups, consist of1 to 12 saturated and/or unsaturated carbons, including, but are notlimited to, methyl, ethyl, propyl, butyl, and the like.

As used herein, “aryl” means a monovalent aromatic hydrocarbon radicalof 6 to 20 carbon atoms derived by the removal of one hydrogen atom froma single carbon atom of a parent aromatic ring system. Typical arylgroups, which include, for example, cycloaryl and heteroaryl groups, canbe substituted or non-substituted and include, but are not limited to,radicals derived from benzene, substituted benzene, naphthalene,anthracene, biphenyl, and the like.

In another embodiment, the polynucleotide sequence comprises a 5′phosphorothiolate linkage and is represented by the general structure[IV]:

A modified polynucleotide sequence comprising a 5′ phosphorothiolatelinkage can be generated by incorporating one or more 5′thiol-nucleoside triphosphates into a growing polynucleotide strand. Asused herein, a “5′ thiol-nucleoside triphosphate” refers to a moleculehaving the general structure [III]:

A “nucleoside” comprises a nitrogenous base linked to a sugar molecule.As used herein, the term includes natural nucleosides in their 2′-deoxyand 2′-hydroxyl forms as described in Kornberg and Baker, DNAReplication, 2nd Ed. (Freeman, San Francisco, 1992) and nucleosideanalogs. For example, natural nucleosides include adenosine, thymidine,guanosine, cytidine, uridine, inosine, deoxyadenosine, deoxythymidine,deoxyguanosine, and deoxycytidine. Nucleoside “analogs” refers tosynthetic nucleosides having modified base moieties and/or modifiedsugar moieties, e.g., as described generally by Scheit, NucleotideAnalogs (John Wiley, New York, 1980). Such analogs include syntheticnucleosides designed to enhance binding properties, reduce degeneracy,increase specificity, and the like. Nucleoside analogs include2-aminoadenosine, 2-thiothymidine, pyrrolo-pyrimidine, 3-methyladenosine, C5-propynylcytidine, C5-propynyluridine, C5-bromouridine,C5-fluorouridine, C5-iodouridine, C5-methylcytidine, 7-deazaadenosine,7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine,2-thiocytidine, etc. Nucleoside analogs may comprise any of theuniversal bases mentioned herein.

As used herein, the term “base” refers to the heterocyclic nitrogenousbase of a nucleotide or nucleotide analog (e.g., a purine, a pyrimidine,a 7-deazapurine). Suitable bases for use in the methods of the inventioninclude, but are not limited to, adenine, cytosine, guanine, thymine,uracil, hypoxanthine and 7-deaza-guanine. These and other suitable baseswill permit a nucleotide bearing the base to beenzymatically-incorporated into a polynucleotide chain or sequence. Thebase will also be capable of forming a base pair involving hydrogenbonding with a base on another nucleotide or nucleotide analog. The basepair can be either a conventional (standard) Watson-Crick base pair or anon-conventional (non-standard) non-Watson-Crick base pair, for example,a Hoogstein base pair or bidentate base pair.

As used herein, “Watson-Crick base pair” refers to a pair ofhydrogen-bonded bases on opposite antiparallel strands of a nucleicacid. The rules of base pairing, which were first elaborated by Watsonand Crick, are well known to those of skill in the art. For example,these rules require that adenine (A) pairs with thymine (T) or uracil(U), and guanine (G) pairs with cytosine (C), with the complementarystrands anti-parallel to one another. As used herein, the term“Watson-Crick base pair” encompasses not only the standard AT, AU or GCbase pairs, but also base pairs formed between non-standard or modifiedbases of nucleotide analogs capable of hydrogen bonding to a standardbase or to another complementary non-standard base. One example of suchnon-standard Watson-Crick base pairing is the base pairing whichinvolves the nucleotide analog inosine, wherein its hypoxanthine baseforms two hydrogen bonds with adenine, cytosine or uracil of othernucleotides.

As used herein, the term “polynucleotide sequence” refers to a nucleicacid molecule (e.g., DNA, RNA) that is produced by the incorporation oftwo or more nucleoside triphosphates into a single molecule via one ormore covalent linkages (e.g., a phosphodiester bond, a phosphorothiolatelinkage). A “template polynucleotide sequence” can be any nucleotidesequence for which it is desirable to produce or to obtain sequenceinformation using the methods described herein. The templatepolynucleotide sequence may be a polynucleotide sequence (e.g.,oligonucleotide sequence) and may be single-stranded or double-stranded.A template that is initially provided in double-stranded form can betreated to separate the two strands (e.g., the DNA will be denatured).The template polynucleotide also may be naturally-occurring, isolated orsynthetic. Examples of suitable templates include, but are not limitedto, genomic DNA, mitochondrial DNA, complementary DNA (cDNA), a PCRproduct and other amplified nucleotides. RNA may also be used as atemplate. For example, RNA can be reverse transcribed to yield cDNA,using methods known in the art such as RT-PCR. The templatepolynucleotide sequence may be used in any convenient form, according totechniques known in the art (e.g., isolated, cloned, amplified), and maybe prepared for the sequencing reaction, as desired, according totechniques known in the art. In a particular embodiment, the templatepolynucleotide sequence comprises DNA. In a further embodiment, thetemplate polynucleotide sequence comprises a sense DNA strand and anantisense DNA strand, wherein at least one primer is annealed to atleast one strand (e.g., sense strand, antisense strand) or to both senseand antisense strands.

Template polynucleotides can be obtained from any of a variety ofsources. For example, DNA may be isolated from a sample, which may beobtained or derived from a subject. The word “sample” is used in a broadsense to denote any source of a template on which sequence determinationis to be performed. The source of a sample may be of any viral,prokaryotic, archaebacterial, or eukaryotic species. The sample may beblood or another bodily fluid containing cells; sperm; and a biopsy(e.g., tissue) sample, among others.

As used herein, the term “primer” refers to an oligonucleotide, which iscomplementary to the template polynucleotide sequence and is capable ofacting as a point for the initiation of synthesis of a primer extensionproduct. In one embodiment, the primer is complementary to the sensestrand of a polynucleotide sequence and acts as a point of initiationfor synthesis of a forward extension product. In another embodiment, theprimer is complementary to the antisense strand of a polynucleotidesequence and acts as a point of initiation for synthesis of a reverseextension product.

The primer may occur naturally, as in a purified restriction digest, orbe produced synthetically. The appropriate length of a primer depends onthe intended use of the primer, but typically ranges from about 5 toabout 200; from about 5 to about 100; from about 5 to about 75; fromabout 5 to about 50; from about 10 to about 35; from about 18 to about22 nucleotides. A primer need not reflect the exact sequence of thetemplate but must be sufficiently complementary to hybridize with atemplate for primer elongation to occur, i.e., the primer issufficiently complementary to the template polynucleotide sequence suchthat the primer will anneal to the template under conditions that permitprimer extension. As used herein, the phrase “conditions that permitprimer extension” refers to those conditions, e.g., salt concentration(metallic and non-metallic salts), pH, temperature, and necessarycofactor concentration, among others, under which a given polymeraseenzyme catalyzes the extension of an annealed primer. Conditions for theprimer extension activity of a wide range of polymerase enzymes areknown in the art. As one example, conditions permitting the extension ofa nucleic acid primer by Taq polymerase include the following (for anygiven enzyme, there can and often will be more than one set of suchconditions): reactions are conducted in a buffer containing 50 mM KCl,10 mM Tris (pH 8.3), 4 mM MgCl₂, (200 μM of one or more dNTPs and/or achain terminator may be included, depending upon the type of primerextension or sequencing being performed); reactions are performed at 72°C.

It will be clear to persons skilled in the art that the size of theprimer and the stability of hybridization will be dependent to somedegree on the ratio of A-T to C-G base pairings, since more hydrogenbonding is available in a C-G pairing. Also, the skilled person willconsider the degree of homology between the extension primer to otherparts of the amplified sequence and choose the degree of stringencyaccordingly. Guidance for such routine experimentation can be found inthe literature, for example, Molecular Cloning: A Laboratory Manual bySambrook, J., Fritsch E. F. and Maniatis, T. (1989).

In the methods of the present invention, tags can be used to facilitatethe production and/or sequencing of polypeptide sequences. As usedherein, a “tag” or “label” are used interchangeably to refer to anymoiety that is capable of being specifically detected (e.g., by bindingto an isolating means (e.g., a partner moiety)), either directly orindirectly, and therefore, can be used to identify and/or isolate apolynucleotide sequence that comprises the tag. Suitable tags for use inthe methods of the present invention can be present on a primer, amodified polynucleotide sequence, a template, or on one or morenucleoside triphosphates (e.g., non-modified or standard nucleosidetriphosphates, modified nucleoside triphosphates) and include, amongothers, affinity tags (e.g., biotin, avidin, streptavidin), haptens,ligands, peptides, nucleic acids, fluorophores, chromophores, andepitope tags that are recognized by an antibody (e.g., digoxigenin(DIG), hemagglutinin (HA), myc, FLAG) (Andrus, A. “Chemical methods for5′ non-isotopic labelling of PCR probes and primers” (1995) in PCR 2: APractical Approach, Oxford University Press, Oxford, pp. 39-54). Othersuitable tags include, but are not limited to, chromophores,fluorophores, haptens, radionuclides (e.g., ³²P, ³³P, ³⁵S), fluorescencequenchers, enzymes, enzyme substrates, affinity tags (e.g., biotin,avidin, streptavidin, etc.), mass tags, electrophoretic tags and epitopetags that are recognized by an antibody. In certain embodiments, thelabel is present on the 5 carbon position of a pyrimidine base or on the3 carbon deaza position of a purine base. In a particular embodiment,the primer comprises at least one tag or label.

In a further embodiment, the primer comprises at least one affinity tag.As defined herein, an “affinity tag” refers to a moiety that can beattached to a nucleoside or nucleoside analog, which can bespecifically-bound by a partner moiety. The interaction of the affinitytag and its partner permits the isolation (i.e., specific capture andpurification) of molecules bearing the affinity tag. Suitable examplesinclude, but are not limited to, biotin or iminobiotin and avidin orstreptavidin. A sub-class of affinity tag is the “epitope tag,” whichrefers to a tag that is recognized and specifically bound by an antibodyor an antigen-binding fragment thereof. Examples of epitope tags includethe Myc tag, recognized by monoclonal anti-Myc antibodies; FLAG™ tag,recognized by anti-FLAG™ antibodies; and digoxigenin, recognized byanti-digoxigenin antibodies. In one embodiment, the primer comprises abiotin tag. In another embodiment, the primer comprises a digoxigenintag.

In another embodiment, the primer comprises a tag (e.g., a label) thatis a fluorophore. Suitable fluorophores can be provided as fluorescentdyes, including, but not limited to Alexa Fluor dyes (Alexa Fluor 350,Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568,Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660 and Alexa Fluor 680),AMCA, AMCA-S, BODIPY dyes (BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR,BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY581/591, BODIPY 630/650, BODIPY 650/665), CAL dyes, Carboxyrhodamine 6G,carboxy-X-rhodamine (ROX), Cascade Blue, Cascade Yellow, Cyanine dyes(Cy3, Cy5, Cy3.5, Cy5.5), Dansyl, Dapoxyl, Dialkylaminocoumarin,4′,5′-Dichloro-2′,7′-dimethoxy-fluorescein, DM-NERF, Eosin, Erythrosin,Fluorescein, Carboxy-fluorescein (FAM), Hydroxycoumarin, IRDyes (IRD40,IRD 700, IRD 800), JOE, Lissamine rhodamine B, Marina Blue,Methoxycoumarin, Naphthofluorescein, Oregon Green 488, Oregon Green 500,Oregon Green 514, Oyster dyes, Pacific Blue, PyMPO, Pyrene, Rhodamine6G, Rhodamine Green, Rhodamine Red, Rhodol Green,2′,4′,5′,7′-Tetra-bromosulfone-fluorescein, Tetramethyl-rhodamine (TMR),Carboxytetramethylrhodamine (TAMRA), Texas Red, and Texas Red-X. Inother embodiments, the label is a mass tag or an electrophoretic tag.

In addition to the various detectable moieties mentioned above, thepresent invention also comprehends the use of tags or labels, such asspectrally resolvable quantum dots, metal nanoparticles or nanoclusters,etc., which may either be directly attached to an oligonucleotide primeror may be embedded in or associated with a polymeric matrix which isthen attached to the primer. As mentioned above, detectable moietiesneed not themselves be directly detectable. For example, they may act ona substrate which is detected, or they may require modification tobecome detectable.

As described herein, the primers and nucleotide triphosphates cancomprise one or more tags or labels (e.g., a first tag, second tag,third tag, fourth tag, fifth tag, sixth tag, seventh tag, eighth tag).The various tags used in the methods can be the same or different(distinct). In a particular embodiment, when more than one tag is usedin the method, the tags (e.g., first, second) are different (distinct),such that the primers or nucleoside triphosphates can be separated fromone another using the distinct tag or tags.

As will be appreciated by one of ordinary skill in the art, referencesto templates, primers, etc., generally mean populations or pools ofnucleic acid molecules that are substantially identical within arelevant region rather than single molecules. Thus, for example, a“template” generally means a plurality of substantially identicaltemplate molecules.

According to the methods of the invention, primer extension is carriedout in the presence of one or more nucleoside triphosphates. In oneembodiment, the nucleoside triphosphates are a mixture of standarddeoxynucleo side triphosphates (also referred to herein as dNTPs (e.g.,dATP, dCTP, dGTP, dTTP, dUTP) and pppNs (e.g., pppA, pppC, pppG, pppT))and modified thiol-deoxynucleoside triphosphates (e.g., 3′ and 5′thiol-nucleoside triphosphates (also referred to herein as sdNTPs (e.g.,sdATP, sdCTP, sdGTP, sdTTP, sdUTP) and dNsTPs (e.g., dAsTP, dCsTP,dGsTP, dTsTP, dUsTP))). In a particular embodiment, the mixture ofnucleoside triphosphates comprises four standard deoxynucleotidetriphosphates and one or more thiol-nucleoside triphosphates. Suitablethiol-nucleoside triphosphates include, but are not limited to, thosewhich comprise a base that is either adenine, cytosine, thymine, guanineor uracil. In particular embodiments of the methods of the invention,each standard dNTP is present at a higher concentration than itscorresponding modified dNTP (e.g, dCTP is present in the mixture at ahigher concentration than the modified thiol-dCTP in the same mixture)so that the modified dNTP will be incorporated less frequently than thestandard dNTP, as will be understood by the person of skill in the art.

The modified thiol-nucleoside triphosphates in the mixture of nucleosidetriphosphates can be unlabeled or can comprise one or more labels, asdescribed herein.

In a particular embodiment, the mixture of nucleotides comprises fourlabeled thiol-nucleoside triphosphates (e.g., sdCTP, sdTTP, sdATP, andsdGTP). In a particular embodiment, each of the four thiol-nucleosidetriphosphates comprises a distinct label, which is not present on anyother nucleotide in the mixture. Thus, in certain embodiments, the labelis present on the 5 carbon position of a pyrimidine base or on the 7carbon deaza position of a purine base. A person of skill in the artwill recognize that a polynucleotide sequence can be determined,according to the methods of the present invention, by performing asingle reaction that utilizes a mixture of four conventional nucleosidetriphosphates and four modified 3′ thiol-nucleoside triphosphates,wherein each of the four modified 3′ thiol-nucleoside triphosphatescomprises a distinct label that is not present on any other nucleotidein the mixture.

Extension of a primer (e.g., DNA synthesis) can be accomplished using anucleic acid polymerase which is capable of enzymatically-incorporatingboth standard (dNTPs) and modified thiol deoxynucleotides (sdNTPs) intoa growing nucleic acid strand. As used herein, the phrase “nucleic acidpolymerase enzyme” refers to an enzyme (e.g., naturally-occurring,recombinant, synthetic) that catalyzes the template-dependentpolymerization of nucleoside triphosphates to form primer extensionproducts that are complementary to one of the nucleic acid strands ofthe template nucleic acid sequence. Numerous nucleic acid polymerasesare known in the art and are commercially available. Nucleic acidpolymerases that are thermostable, i.e., they retain function afterbeing subjected to temperatures sufficient to denature annealed strandsof complementary nucleic acids, are particularly useful for the methodsof the present invention.

Suitable polymerases for the methods of the present invention includeany polymerase known in the art to be useful for recognizing andincorporating standard deoxynucleotides. Examples of such polymerasesare disclosed in Table 1 of U.S. Pat. No. 6,858,393, the contents ofwhich are incorporated herein by reference. Many polymerases are knownby those of skill in the art to possess a proof-reading, orexonucleolytic activity, which can result in digestion of 3′ ends thatare available for primer extension. In order to avoid this potentialproblem, it may be desirable to use polymerase enzyme which lack thisactivity (e.g., exonuclease-deficient polymerases, referred to herein asexo-polymerases). Such polymerases are well known to those of skill inthe art and include, for example, Klenow fragment of E. Coli DNApolymerase I, Sequenase, exo-Thermus aquaticus (Taq) DNA polymerase andexo-Bacillus stearothermophilus (Bst) DNA polymerase. In a particularembodiment, incorporation of modified thiol deoxynucleotides (sdNTPs)into DNA is accomplished using a DNA amplification reaction, such asPCR. Therefore, especially suitable polymerases for the methods of thepresent invention include those that are stable and function at hightemperatures (i.e., thermostable polymerases useful in PCR thermalcycling). Examples of such polymerases include, but are not limited to,Thermus aquaticus (Taq) DNA polymerase, TaqFS DNA polymerase,thermosequenase, Therminator DNA polymerase, Tth DNA polymerase, Pfu DNApolymerase and Vent (exo-)DNA polymerase. In another embodiment,incorporation of modified thiol-nucleoside triphosphates into RNA isaccomplished using an RNA polymerase. Examples of RNA polymerasesinclude, but are not limited to, E. coli RNA polymerase, T7 RNApolymerase and T3 RNA polymerases.

The present invention also encompasses a method for determining all or aportion of a polynucleotide sequence comprising: annealing a pluralityof primers to a plurality of template polynucleotide sequences; andextending the plurality of the primers in the presence of one or morenucleoside triphosphates wherein at least one of the nucleosidetriphosphates is modified, thereby producing a plurality of extensionproducts that comprise a modified nucleotide sequence having one or morephosphorothiolate linkages. The phosphorothiolate linkages in theextension products are cleaved under conditions in which a plurality offragments are produced; and the fragments of the of the extensionproducts that comprise a primer are then identified (e.g., using tags,labels, solid supports and other means described herein), and thenucleotide at the 3′ end of the fragments is subsequently identified,such that the polynucleotide sequence can be determined.

In one embodiment, the at least one modified nucleotide comprises ageneral structure [I]:

such that the modified nucleotide sequences that are produced comprise ageneral structure [II]:

wherein the general structure [II] comprises at least one 3′phosphorothiolate linkage.

In another embodiment, the at least one modified nucleotide comprises ageneral structure [III]:

such that the modified nucleotide sequences that are produced comprise ageneral structure [IV]:

wherein the general structure [IV] comprises at least one 5′phosphorothiolate linkage.

One of skill in the art will recognize that, in order to determine thesequence of a polynucleotide using the methods of the present invention,a ladder of fragments in which each fragment comprises a primer can beproduced by cleavage of a plurality of extension products. One of skillin the art will appreciate that four separate extension reactions can beperformed in which a different modified dNTP (e.g., thiol-nucleosidetriphosphate) is used in each of the four reactions. Upon cleaving theextension products to produce fragments of various sizes and resolvingthe fragments (e.g., on a gel), four distinct ladders are produced,wherein each fragment in an individual ladder has at its 3′ end the samemodified nucleotide that was used for the extension reaction. Bydetermining the size of each fragment that has a known nucleotide at its3′ end and comparing the size of the fragments in the four individualladders, the sequence of the extension product can be determined. Oncethe sequence of the extension product is known, the sequence of thetemplate polynucleotide, which is the reverse complement of the sequenceof the extension product, can be determined.

Alternatively, one of skill in the art will recognize that a singleextension reaction comprising 4 modified dNTPs (e.g., thiol-nucleosidetriphosphates), wherein each reaction comprises four distinct labelscorresponding to the four bases (e.g., a distinct label on more than onemodified nucleoside triphosphate, more than one dNTP, more than oneprimer, more than one etc., and combinations thereof), wherein eachdistinct label can be used to generate a single sequence ladderrepresenting the different bases. Thus, the ladder comprises fragmentsthat represent the full-length extension product and various 3′truncations thereof. Preferably, all possible 3′ truncations of theextension product are produced, such that the complete sequence of thepolynucleotide can be determined. By resolving the ladder of fragments(e.g., on a gel), identifying the nucleotide at the 3′ end of eachfragment (e.g., using the distinct label or tag on the nucleotide, suchas a fluorophore) and reading the sequence ladder (e.g., on a gel),beginning with the nucleotide at the 3′ end of the smallest fragment andending with the nucleotide at the 3′ end of the largest fragment, thesequence of the polynucleotide can be determined. Once the sequence ofthe extension product is known, the sequence of the templatepolynucleotide, which is the reverse complement of the sequence of theextension product, can be determined.

As used herein, the phrase “determining a polynucleotide sequence”,“sequencing”, and like terms, in reference to polynucleotides, includesdetermination of partial as well as full sequence information of thepolynucleotide. That is, the term includes sequence comparisons,fingerprinting, and like levels of information about a targetpolynucleotide, as well as the express identification and ordering ofeach nucleoside of the target polynucleotide within a region ofinterest. In certain embodiments of the invention “determining apolynucleotide sequence” comprises identifying a single nucleotide,while in other embodiments more than one nucleotide is identified. Incertain embodiments of the invention, sequence information that isinsufficient by itself to identify any nucleotide in a single cycle isgathered. Identification of nucleosides, nucleotides, and/or bases areconsidered equivalent herein. It is noted that performing sequencedetermination on a polynucleotide typically yields equivalentinformation regarding the sequence of a perfectly complementary (100%complementary) polynucleotide, and thus, is equivalent to sequencedetermination performed directly on a perfectly complementarypolynucleotide. The methods described herein allow partial determinationof a sequence, e.g., the identification of individual nucleotides spacedapart from one another in a template. In certain embodiments of theinvention, in order to gather more complete information, a plurality ofreactions is performed.

In one embodiment of the invention, the identity of one or morenucleotides is determined using the methods described herein, for thepurpose of detecting a polymorphism. The term “polymorphism” is givenits ordinary meaning in the art and refers to a difference in anucleotide sequence (e.g., genomic sequence) among individuals (e.g., ofthe same species). In a particular embodiment, the polymorphism is a“single nucleotide polymorphism” (SNP), which refers to a polymorphismat a single position. In other embodiments of the invention, the methodsfor determining a polynucleotide sequence are employed to determine theidentity of multiple nucleotides (e.g., more than one) in a templatepolynucleotide sequence.

In particular embodiments, a plurality of extension products thatcomprises a modified nucleotide sequence having one or morephosphorothiolate linkages are produced using polymerase chain reaction(PCR). Methods for performing PCR are well known in the art and aredescribed, for example, in U.S. Pat. Nos. 4,683,195, 4,683,202, and4,965,188, and in Dieffenbach, C. and Dveksler, G S, PCR Primer: ALaboratory Manual, 2^(nd) ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, 2003.

The methods for determining a polynucleotide sequence comprise cleavingphosphorothiolate linkages in the extension products under conditions inwhich a plurality of fragments of the modified nucleotide sequenceattached to a plurality of primers are produced. One of skill in the artwill recognize that such conditions can also result in the separation ofthe cleaved fragments from the template polynucleotide sequence to whichthey are annealed, thereby producing a single-stranded polynucleotide. Aphosphorothiolate linkage (3′ or 5′) in a polynucleotide sequence can beefficiently cleaved according to methods known in the art (Vyle, J. S.,et al., Biochemistry 31: 3012-3018 (1992); Sontheimer, E. J., et al.,Methods 18: 29-37 (1999); Mag, M., et al., Nucleic Acids Res.,19(7):1437-1441 (1991)). For example, cleavage of a phosphorothiolatelinkage can be accomplished chemically, by exposing (e.g., contacting)the polynucleotide sequence to certain metal agents. The metal can be,for example, silver (Ag), mercury (Hg), copper (Cu), manganese (Mn),zinc (Zn) or cadmium (Cd), among others. Water-soluble salts thatprovide Ag⁺, Hg⁺⁺, Cu⁺⁺, Mn⁺⁺, Zn⁺ or Cd⁺ anions (salts that provideions of other oxidation states can also be used) are particularlyuseful. Iodide (I₂) can also be used. Silver-containing salts such assilver nitrate (AgNO₃), or other salts that provide Ag⁺ ions, areparticularly useful in the methods of the present invention.

Suitable conditions for cleaving a phosphorothiolate linkage present ina polynucleotide sequence include, but are not limited to, incubatingthe polynucleotide sequence with a metal agent, such as Ag⁺ ions, at apH in the range of from about 4.0 to about 10.0, from about 5.0 to about9.0 or from about 6.0 to about 8.0, and at a temperature in the range offrom about 15° C. to about 50° C., from about 20° C. to about 45° C.,from about 25° C. to about 40° C., from about 22° C. to about 37° C., orfrom about 24° C. to about 32° C. Particular suitable conditionsinclude, for example, incubation in the presence of 50 mM AgNO₃ at about22 to about 37° C. for at least about 10 minutes at a pH of about 7.0.An example of conditions for a cleavage reaction are described inExample 2. Such conditions can optionally comprise an additional step inwhich the cleaved fragments are separated from the templatepolynucleotide sequence to which they are annealed (e.g., incubation ata temperature from about 90° C. to about 100° C. for about 30 seconds toabout 60 seconds) prior to or at the same time the phosphorothiolatelinkages are cleaved.

In a further embodiment of the invention, the method for determining apolynucleotide sequence additionally comprises isolating a cleavedfragment (e.g., using an isolating means) subsequent to the cleavagereaction. In a particular embodiment, the cleaved fragment that isisolated comprises the primer that was extended in the extensionreaction. As used herein, the term “isolated fragment” refers to apreparation of fragments that is purified from, or otherwisesubstantially free of, other components from the extension and/orcleavage reactions, including, but not limited to, cleavage fragmentsthat are not attached to a primer, buffers, unincorporated nucleotides,nucleic acid templates and enzymes. Such fragments can be isolated usingan isolating means, for example, a support (e.g., magnetic beads,agarose or sepharose beads, among others) that comprises a moiety whichrecognizes and binds to a tag (e.g., a tag on a primer). Examples ofpairs of partner moieties that are suitable for the present inventioninclude, but are not limited to, biotin and streptavidin/avidin, or anepitope (e.g., digoxigenin (DIG)) and an antibody that recognizes andbinds the epitope (e.g., an anti-DIG antibody).

“Support”, as used herein, refers to a matrix on or in which nucleicacid molecules, microparticles, and the like may be immobilized, e.g.,to which they may be covalently or noncovalently attached or, in or onwhich they may be partially or completely embedded so that they arelargely or entirely prevented from diffusing freely or moving withrespect to one another. The term “microparticle” is used herein to referto particles having a smallest cross-sectional dimension of 50 micronsor less, preferably 10 microns or less. Microparticles may be made of avariety of inorganic or organic materials including, but not limited to,glass (e.g., controlled pore glass), silica, zirconia, cross-linkedpolystyrene, polyacrylate, poly-methylmethacrylate, titanium dioxide,latex, polystyrene, etc. See, e.g., U.S. Pat. No. 6,406,848, for varioussuitable materials and other considerations. Magnetically responsivemicroparticles can be used.

The magnetic responsiveness of certain preferred microparticles permitsfacile collection and concentration of the microparticle-attachedtemplates after amplification, and facilitates additional steps (e.g.,washes, reagent removal, etc.). In certain embodiments of the inventiona population of microparticles having different shapes (e.g., somespherical and others nonspherical) is employed. In general, any pair ofmolecules that exhibit affinity for one another such that they form abinding pair may be used to attach microparticles or templates to asubstrate. The first member of the binding pair is attached covalentlyor noncovalently to the substrate, and the second member of the bindingpair is attached covalently or noncovalently to the microparticles ortemplates.

In other embodiments of the invention, the templates are amplified bypolymerase chain reaction (PCR) in a semi-solid support, such as a gelhaving suitable amplification primers immobilized therein. Templates,additional amplification primers, and reagents needed for the PCRreaction are present within the semi-solid support. One or both of apair of amplification primers is attached to the semi-solid support viaa suitable linking moiety, e.g., an acrydite group. Attachment may occurduring polymerization. Additional reagents (e.g., templates, secondamplification primer, polymerase, nucleotides, cofactors, etc.) may bepresent prior to formation of the semi-solid support (e.g., in a liquidprior to gel formation), or one or more of the reagents may be diffusedinto the semi-solid support after its formation. The pore size of thesemi-solid support is selected to allow such diffusion. As is well knownin the art, in the case of a polyacrylamide gel, pore size is determinedmainly by the concentration of acrylamide monomer and to a lesser extentby the crosslinking agent. Similar considerations apply in the case ofother semi-solid support materials. Appropriate cross-linkers andconcentrations to achieve a desired pore size can be selected.

In certain embodiments of the invention an additive such as a cationiclipid, polyamine, polycation, etc., is included in the solution prior topolymerization, which forms in-gel micelles or aggregates surroundingthe microparticles. Methods disclosed in U.S. Pat. Nos. 5,705,628,5,898,071, and 6,534,262 and U.S. Patent Application Publication No.2002/0106686, each of which are incorporated herein by reference, mayalso be used. For example, various “crowding reagents” can be used tocrowd DNA near beads for clonal PCR. SPRI® magnetic bead technologyand/or conditions can also be employed. See, e.g., U.S. Pat. No.5,665,572, demonstrating effective PCR amplification in the presence of10% polyethylene glycol (PEG). In certain embodiments of the inventivemethods amplification (e.g., PCR), ligation, or both, are performed inthe presence of a reagent such as betaine, polyethylene glycol, PVP-40,or the like. These reagents may be added to a solution, present in anemulsion, and/or diffused into a semi-solid support.

Numerous other supports are known in the art, some of which aredescribed in U.S. Pat. No. 6,828,100, the contents of which are hereinincorporated by reference. In general, any of a wide variety of methodsknown in the art can be used to modify nucleic acids such asoligonucleotide primers, probes, templates, etc., to facilitate theattachment of such nucleic acids to microparticles or to other supportsor substrates.

As will be understood by a person of skill in the art, isolatedextension products can be identified, either directly or indirectly,using one of many standard and well-known detection methods and/ortechniques. Such methods and/or techniques include, but are not limitedto, fluorescence detection, spectrophotometric detection, chemicaldetection and/or electrophoretic detection. In one embodiment, detectionof isolated extension products is accomplished by resolving the primerextension products by means of, for example, high-resolution denaturingpolyacrylamide/urea gel electrophoresis, capillary separation, or otherresolving means; followed by detecting the fragments, for example, usinga scanning spectrophotometer or fluorometer. In a particular embodiment,fluorescently-labeled primer extension products are resolved by gelelectrophoresis, according to procedures that are well known in the art,and are subsequently detected in the gel using a standard fluorometer.

Electrophoretic separation of the isolated cleavage fragments produces a“ladder” of extension fragments, each fragment starting with the primerand ending with one of the four modified thiol-nucleotides at its 3′end. The sequence of the complement (i.e., the primer extensionproduct), from which the sequence of the template can be deduced, isread directly from the order of fragments on the gel.

Techniques for detecting nucleic acid fragments on a gel are well knownin the art. Furthermore, one of skill in the art will recognize that theparticular method of detection will depend on the specific labelcomprised by the resolved fragments. For example, if the fragments arelabeled with a fluorophore, then standard fluorescence-based techniquescan be utilized to detect the fragments in a gel.

The skilled artisan will recognize that a polynucleotide sequence can bedetermined, according to the methods of the present invention, byperforming a single reaction that utilizes a mixture of fourconventional nucleoside triphosphates and four modified 3′thiol-nucleoside triphosphates, wherein each of the four modified 3′thiol-nucleoside triphosphates comprises a distinct label that is notpresent on any other nucleotide in the mixture (e.g., four colorsequencing).

Alternatively, one of skill in the art will also recognize that apolynucleotide sequence can be determined, according to the methods ofthe present invention, by performing four separate reactions todetermine the nucleotide sequence of a template when 5′ thiol-nucleosidetriphosphates are utilized to generate modified polynucleotide sequencescomprising one or more 5′ phosphorothiolate linkages. In a particularembodiment, each of the four reactions comprises four conventionalnucleoside triphosphates and only one of four modified 5′thiol-nucleoside triphosphates, for example, sdCTP, sdATP, sdGTP, orsdTTP. When four reactions are performed using the same templatepolynucleotide sequence, each with one of the four modified 5′thiol-nucleoside triphosphates, for example, sdCTP, sdATP, sdGTP orsdTTP, the products of the reactions can be cleaved and detected,according to the methods described herein, and analyzed to determine thesequence of the template polynucleotide (see, for example, Sanger, F.,et al. Proc. Natl. Acad. Sci. USA 74: 5463-5467 (1977) and Maxam, A. M.and Gilbert, W. Proc. Natl. Acad. Sci. USA 74: 560-564 (1977), thecontents of each are incorporated by reference herein).

In other embodiments of the invention, the methods described herein canbe performed using a template polynucleotide sequence comprising a senseand antisense nucleotide strand and two primers, a forward primer and areverse primer, such that sequence information for both the sense andantisense strands of the template polynucleotide sequence can bedetermined. In one embodiment, each primer comprises at least onedistinct tag that is not present on the other primer. After primerextension is performed to produce extension products that comprisemodified polynucleotide sequences having one or more phosphorothiolatelinkages, followed by cleavage of the phosphorothiolate linkages in themodified polynucleotide sequences—both performed according to methodsdescribed herein—two populations of cleaved extension fragments can beisolated, also according to methods described herein. The firstpopulation consists of cleavage fragments, each of which comprises theforward primer, while the second population consists of cleavagefragments, each of which comprises the reverse primer. These populationscan be separated from one another and the sequences of the forward andreverse extension products can be determined, as described herein, suchthat the sequences of both the antisense and sense strands of thetemplate polynucleotide sequence can be determined.

Accordingly, the present invention also provides a method for separatingone or more forward extension products from one or more reverseextension products comprising annealing a plurality of first primers anda plurality of second primers to a plurality of template polynucleotidesequences comprising a sense nucleotide strand and an antisensenucleotide strand, wherein the first primer anneals to the sense strandand the second primer anneals to the antisense strand and wherein atleast one primer comprises a tag. The first and second primers areextended in the presence of one or more nucleoside triphosphates,wherein at least one of the nucleoside triphosphates is modified,thereby producing a plurality of extension products that comprise amodified nucleotide sequence having one or more phosphorothiolatelinkages. The phosphorothiolate linkages in the modified extensionproducts are cleaved under conditions in which a plurality of fragmentsare produced; and the fragments attached to the first primers areseparated from the fragments attached to the second primers.

In a particular embodiment, the first primer and the second primer eachcomprise a tag, wherein the tag on the first primer is distinct from thetag on the second primer. Accordingly, the fragments of the reverseextension product that comprise the first primer can be separated fromthe fragments of the forward extension product that comprise the secondprimer using the distinct tags on the first and second primers. Thefragments of the forward and reverse extension products can beidentified either simultaneously or in succession.

In another embodiment, two primers, a forward and reverse primer, areused to amplify the template polynucleotide sequence by polymerase chainreaction (PCR), according to procedures that are well known in the art.Forward and reverse primer extension products with at least onephosphorothiolate linkage, whose sequences correspond to the sense andantisense strands of the template sequence, respectively, are generated.These products can then be cleaved, isolated and detected, according tomethods described herein, in order to deduce the sequence of thetemplate polynucleotide.

The invention also encompasses a kit, which can comprise one or moremodified thiol-nucleoside triphosphates (sdNTPs), conventionalnucleoside triphosphates (dNTPs) and/or a nucleic acid polymerase (e.g.,Klenow fragment of E. Coli DNA polymerase I, Sequenase, exo-Thermusaquaticus (Taq) DNA polymerase and exo-Bacillus stearothermophilus (Bst)DNA polymerase). The modified sdNTPs can be either labeled or unlabeled.In a particular embodiment, the sdNTPs comprise a fluorescent label(e.g., a fluorophore). In certain embodiments, the detectable label ispresent on the 5 carbon position of a pyrimidine base or on the 7 carbondeaza position of a purine base. In another embodiment, the standard andmodified nucleotides comprise a base, such as, but not limited to,adenine, guanine, cytosine, thymine, uracil, hypoxanthine or7-deaza-guanine. Such kits can be used, for example, to produce and/ordetermine the sequence of a modified polynucleotide that comprises a(e.g., one or more) phosphorothiolate linkage.

The modified thiol nucleotides can be either 3′ thiol nucleotides or 5′thiol nucleotides. In a particular embodiment the 3′ thiol nucleotidescomprise either a 3′ thiol group (—SH) or a 3′ dithiomethyl group(—SSCH₃), for example, 3′-deoxy-dithiomethyl thymidine (see FIG. 3B). Ina another embodiment, the modified 5′ thiol nucleotides are 5′phosphorothiolate dNTPs.

Other components that are suitable for the kits of the inventioninclude, but are not limited to, an extension buffer (e.g., buffers,salts, magnesium (Mg)), a cleavage buffer, pyrophosphate, one or moresupports for isolating extension products, one or more reagents forsample clean-up (e.g., CleanSeq, AmPure) and manufacturer'sinstructions.

A suitable cleavage buffer comprises a source of one or more metal ions,for example silver, mercury or copper. In a particular embodiment, thecleavage buffer comprises a source of silver ions, such as silvernitrate (AgNO₃) or silver acetate. In a further embodiment, the sourceof silver ions is provided at a concentration in the range of 1-100 mM.Additionally, the cleavage buffer can further comprise a source ofmagnesium ions (Mg⁺⁺). In a particular embodiment, the source ofmagnesium ions is magnesium acetate.

Example 1 Incorporation of 3′ Thiol-Nucleoside Triphosphates into aGrowing Strand of DNA by DNA Polymerase Materials and Methods

DNA synthesis was performed on a single-stranded DNA template (5′-TTTTTT CTA AGG TAG CGA CTG TCC TAT ACA GAC TGA CAA AAA AAG AGA ATG AGG AACCCG GGG CAG-3′) (SEQ ID NO:1), which was labeled at its 5′ end with adual biotin tag and was attached to a magnetic bead (FIG. 3B). Synthesiswas primed using a primer (5′-CTG CCC CGG GTT CCT CAT TCT CT-3′) (SEQ IDNO:2), which was complementary to a portion of the DNA template. Theprimer was labeled with Cy5 at its 5′ end. The DNA template contained 5adenine nucleotides immediately downstream of the primer sequence.Primer extension reactions were performed using 12.5 U exo⁻ E. coliPolymerase I, Klenow fragment (Epicentre) with 500 μm or 50 μm of3′-deoxy-dithiomethyl thymidine (dTsTP) at 37° C. for 4.0 min.

Results

Up to five 3′-deoxy-dithiomethyl thymidine nucleotides were successfullyincorporated into the DNA amplification product following completion ofthe reaction. When present at a low concentration (5.0 μm), DNA productswith 0, 1, 2, 3, 4 or 5 modified thymidine residues were recovered (FIG.3D). The majority of reaction products, however, contained either 0 or 1modified nucleotide. When present at a higher concentration (1.0 mM),the vast majority of reaction products contained 5 modified thymidineresidues (FIG. 3E). Similar results were observed when reactions wereconducted using other polymerases, including Sequenase, exo-Taqpolymerase and exo-Bst polymerase. These data indicate that modified3′-deoxy-dithiomethyl thymidine nucleotides can be readily incorporatedinto a growing DNA strand during synthesis reactions involving DNApolymerase.

Example 2 Chemical Cleavage of DNA Containing 3′ Thiol ModifiedNucleotides in the Presence of Silver Ions

Prior to cleavage, modified nucleotides containing five 3′ thiolmodified thymidine nucleotides on one strand (see Example 1) were washedwith 25 mM Magnesium acetate. Cleavage was induced by incubating theproducts with 10 μl silver nitrate (AgNO₃) at a concentration of either50 μm (FIG. 4B) or 500 μm (FIG. 4D) for 15 min at room temperature.Unbound cleavage fragments were removed by washing in dH₂O, and boundreaction products containing the primer were analyzed using a standardgel shift assay. Both concentrations of AgNO₃ that were tested resultedin cleavage of the DNA product containing 3′ thiol modified thymidinenucleotides (FIG. 4B and FIG. 4D).

Example 3 Prophetic Example of DNA Synthesis by Primer Extension with 3′Thiol-Nucleoside Triphosphates (sdNTPs) and Recovery ofChemically-Cleaved Extension Products

Primer extension on a DNA template is conducted in the presence of bothunmodified nucleoside triphosphates and modified 3′-thiol-nucleosidetriphosphates (FIG. 1A). When the modified nucleoside triphosphates areused at a low concentration, they incorporate randomly into the growingDNA strand next to natural dNTPs at a low frequency (FIG. 1B).Incorporation of these modified nucleotides into the DNA introduces oneor more 3′-phosphorothiolate linkages (FIG. 5) into the DNA strand. Thesulfur-phosphorus bond of a phosphorothiolate linkage are specificallyand rapidly cleaved by exposure to silver ions (Ag⁺). As a consequence,the addition of silver nitrate (AgNO₃) to the reaction ensures that eachstrand containing a phosphorothiolate linkage is cleaved into 2 or morefragments, depending on the number of 3′-thiol nucleotides in the strand(FIG. 1C). If the primers used in the reaction are labeled with anaffinity tag, such as biotin, the 5′-most fragments, which contain theprimer sequence with the affinity tag, are readily isolated usingstreptavidin magnetic beads (FIG. 1D). Once captured, the remaining,untagged fragments are washed away. The purified fragments can beresolved by gel electrophoresis, resulting in a fragmentation ladder. Ifeach of the four 3′ thiol nucleotide triphosphates arefluorescently-labeled with a distinct fluorophore, the products areanalyzed on a standard fluorescence based sequencing instrument to readthe sequence of the strand. Furthermore, if polymerase chain reaction(PCR) is conducted using two primers (F FIG. 2A-FIG. 2B), wherein eachprimer contains a different tag, the 5′-most extension products fromboth strands are recovered separately and the sequence of both strandscould be analyzed (FIG. 2C).

The relevant teachings of all publications cited herein that have notexplicitly been incorporated by reference, are incorporated herein byreference in their entirety. While this invention has been particularlyshown and described with references to particular embodiments thereof,it will be understood by those skilled in the art that various changesin form and details may be made therein without departing from the scopeof the invention encompassed by the appended claim

1.-94. (canceled)
 95. A method of separating one or more forward extension products from one or more reverse extension products comprising: a) annealing a first primer and a second primer to a template polynucleotide sequence comprising a sense nucleotide strand and an antisense nucleotide strand, wherein the first primer anneals to the sense strand and the second primer anneals to the antisense strand and wherein at least one primer comprises a first tag; b) extending the first primer and the second primer in the presence of one or more nucleoside triphosphates wherein at least one of the nucleoside triphosphates is modified, thereby producing at least one forward extension product and at least one reverse extension product, wherein the at least one forward extension product and the at least one reverse extension product comprises a modified nucleotide sequence having one or more phosphorothiolate linkages; c) cleaving the one or more phosphorothiolate linkages in the at least one forward extension product and the at least one reverse extension product under conditions in which a plurality of fragments are produced; d) identifying from among the fragments produced in c), one or more fragments that comprise a first primer and one or more fragments that comprise a second primer; and e) separating the one or more fragments that comprise a first primer from the one or more fragments that comprise a second primer, thereby separating one or more forward extension products from one or more reverse extension products.
 96. The method of claim 95, wherein the one or more fragments that comprise a first primer and the one or more fragments that comprise a second primer are separated using the first tag.
 97. A method of separating one or more forward extension products from one or more reverse extension products comprising: a) annealing a first primer and a second primer to a template polynucleotide sequence comprising a sense nucleotide strand and an antisense nucleotide strand, wherein the first primer anneals to the sense strand and the second primer anneals to the antisense strand and wherein the first primer and the second primer each comprise a tag, wherein the tag on the first primer is distinct from the tag on the second primer; b) extending the first primer and the second primer in the presence of one or more nucleoside triphosphates wherein at least one of the nucleoside triphosphates is modified, thereby producing at least one forward extension product and at least one reverse extension product, wherein the at least one forward extension product and the at least one reverse extension product comprises a modified nucleotide sequence having one or more phosphorothiolate linkages; c) cleaving the one or more phosphorothiolate linkages in the at least one forward extension product and the at least one reverse extension product under conditions in which a plurality of fragments are produced; d) identifying from among the fragments produced in c), one or more fragments that comprise a first primer and one or more fragments that comprise a second primer; and e) separating the one or more fragments that comprise a first primer from the one or more fragments that comprise a second primer, thereby separating one or more forward extension products from one or more reverse extension products. 